Oxamate alleviates silicotic fibrosis in mice by inhibiting senescence of alveolar type Ⅱ epithelial cells
[Background]The senescence of alveolar type Ⅱ epithelial cells is an important driving factor for the progression of silicotic fibrosis,and the regulatory effects of oxamate on the senescence of alveolar type Ⅱ epithelial cells is still unclear.[Objective]To explore whether lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting senescence of alveolar type Ⅱ epithelial cells [Methods]This study was divided into two parts:in vivo experiments and in vitro experiments.In the first part,forty SPF C57BL/6J male mice were randomly divided into four groups with 10 in each group:control group,silicosis model group,low-dose oxamate treatment group,and high-dose oxamate treatment group.The silicotic mouse model was established by intratracheal instillation of 50 μL SiO2 sus-pension(100 mg·mL-1).The treatment models were prepared by intraperitoneal injection of 100 μL oxamate(225 mmol·L-1 and 1125 mmol·L-1).In the second part,induction of MLE-12 mouse alveolar type Ⅱ epithelial cells was conducted with SiO2.The in vitro experimental groups were ① SiO2 induction groups:control group,50 μg·mL-1 SiO2 group,100 μg·mL-1 SiO2 group,and 200 μg·mL-1 SiO2 group,and ②oxamate treatment groups:control group,SiO2 group(100 μg·mL-1),low-dose oxamate(25 mmol·L-1)treatment group,and high-dose oxamate(50 mmol·L-1)treatment group.Pathological morphology of lung tissues was evaluated after hematoxylin-eosin(HE)staining;deposition of collagen in lung tissues was evaluated after sirius red staining;positive co-expression of prosurfactant protein C(Pro-SPC)and β-galactosidase was detected by immunofluorescence staining;positive expression of β-galactosidase in MLE-12 cells was detected by immunofluorescence staining.The protein expression levels of collagen type Ⅰ(CoL Ⅰ),fibronectin1(FN1),hexokinase 2(HK2),pyruvate kinase isozyme type M2(PKM2),lactate dehydrogenase A(LDHA),p-ataxia telangiectasia and Rad3-related kinase(ATR),and cyclin-de-pendent kinase inhibitors p21,and p16 were detected by Western blotting.[Results]Compared with the control group,the protein expression levels of HK2,PKM2,LDHA,p-ATR,p21,and p16 were significantly up-regulated in the silicosis model group and the SiO2-induced MLE-12 cells(P＜0.05).The in vivo studies showed that,compared with the control group,the silicon nodule area,the collagen deposition area,the proportion of β-galactosidase positive cells,and the protein ex-pression levels of CoL I,FN1,LDHA,p-ATR,p21,and p16 were significantly upregulated in the silicosis model group(P＜0.05).Compared with the silicosis model group,the oxamate treatment groups showed significant downregulation of the silicon nodule area,the collagen deposition area,the proportion of β-galactosidase positive cells,and the the CoL I,FN1,LDHA,p-ATR,p21,and p16 protein expression levels,and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group(P＜0.05).The in vitro studies showed that,compared with the control group,the proportion of β-galactosidase positive cells and the protein expression levels of p-ATR,p21,and p16 were significantly upregulated in the SiO2-induced group(P＜0.05).Compared with the SiO2 group,the proportion of β-galactosidase positive cells and the LDHA,p-ATR,p21 and p16 protein expression levels were significantly downregulated in the oxamate treatment groups,and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group(P＜0.05).[Conclusion]Lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting the senescence of alveolar type Ⅱ epithelial cells.
oxamatealveolar type Ⅱ epithelial cellsilicosissenescenceβ-galactosidase
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